What is the difference between these two arguments for log fold change shrinkage?
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m-bihie ▴ 10
@4f002f4c
Last seen 12 months ago
Canada

I noticed two different MA plots when I use different inputs for the contrast argument in the lfcShrinkage() function. Both options worked for me but they produced different plots. Does anyone know why and what the difference entails? I used data from datacamp, so my apologies if someone cannot replicate the issue. I added the code I'm confused about betwen exclamation points.

data source: https://app.datacamp.com/learn/courses/rna-seq-with-bioconductor-in-r

data: https://assets.datacamp.com/production/repositories/1766/datasets/bf1d0eff910f1b2cad36e5acdc2a182e95c63965/fibrosis_smoc2_rawcounts_unordered.csv

Code should be placed in three backticks as shown below

rawcounts <- read_csv("fibrosis_smoc2_rawcounts_unordered.csv")

# Create genotype vector
genotype <- c("smoc2_oe", "smoc2_oe", "smoc2_oe", "smoc2_oe", "smoc2_oe", "smoc2_oe", "smoc2_oe")

# Create condition vector
condition <- c("fibrosis", "fibrosis", "fibrosis", "fibrosis", "normal", "normal", "normal")

# Create data frame
smoc2_metadata <- data.frame(genotype, condition)

rownames(smoc2_metadata) <- c("smoc2_fibrosis1", "smoc2_fibrosis2", "smoc2_fibrosis3", "smoc2_fibrosis4", "smoc2_normal1", "smoc2_normal3", "smoc2_normal4")

#work around to remove geneID column that has "...1" as a name
rawcountsfixed <- rawcounts %>%
  select(-`...1`)
length(rownames(smoc2_metadata))
length(colnames(rawcountsfixed))

#reorder the rows of the metadata to match the order of columns in the counts data
idx <- match(colnames(rawcountsfixed),rownames(smoc2_metadata))
print(idx)

#use the output of the match() function to reorder the rows of the metadata to be in the same order as the columns in the count data
reordered_smoc2_metadata <- smoc2_metadata[idx, ]

# Create DESeq object
dds <- DESeqDataSetFromMatrix(countData = rawcountsfixed,
                             colData = reordered_smoc2_metadata,
                             design = ~ condition)

#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

#results
smoc2res <- results(dds, 
                    contrast = c("condition", #I think this is the factor to
                                 "fibrosis", #factor to compare
                                 "normal"),  #base level
                    alpha = 0.05)

#contrast arguments from datacamp
resNormDC <- lfcShrink(dds = dds, 
                     res = smoc2res, 
                     type = "normal",
                     contrast = c("condition", #I think this is the factor to
                                  "fibrosis", #factor to compare
                                  "normal")  #base level
                    )
# -- plotMA with resNorm - DC way
plotMA(resNormDC)

#second contrast argument 
resNorm2 <- lfcShrink(dds = dds, 
                       res = smoc2res, 
                       type = "normal",
                       coef = 2)



# -- plotMA with resNorm - 2nd way
plotMA(resNorm2)

#!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
sessionInfo( )
DESeq • 554 views
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@mikelove
Last seen 18 hours ago
United States

Can you describe how the plots are different?

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